Transposon insertion sequencing (Tn-Seq) has recently emerged as a powerful next-generation sequencing method that enables querying the contributions of all genes in a bacterial genome toward the fitness of a growing organism. In this method, transposon insertion mutant libraries are constructed and subjected to growth selections. Following selection, the locations of all insertions in the population are counted and can be compared between a control and a target condition, enabling the identification of genes that are both conditionally essential and conditionally detrimental. We have exploited Tn-Seq to probe the basis for the large variations in osmotic and acetate stress tolerance of different laboratory strains of Escherichia coli (K-12 MG1655, BL21(DE3), W, and Crooks). Little is currently known to explain the source of this variation and to enable rational engineering to impart stress tolerance. Tn-Seq revealed many differences and similarities in resistance mechanisms at the genetic level across strains, allowing correlations to be made with growth phenotypes. Cross-strain comparisons of conditionally essential genes and their relative essentiality also suggest a large degree of variation in metabolic flux distributions and regulation of gene expression between strains. A number of direct targets for metabolic engineering of stress resistance via loss-of-function mutations were also discovered, and we show that deletion of a selection of these genes results in improved growth under the original selection condition.